Gene Targeting Outline

 

1. Introduction

This is a brief outline of the steps necessary to produce mice with a mutation targeted to a specific gene. These animals are referred to as “knock-out” mice or “gene targeted” mice. The broad outline of the experiment includes: 1) the investigator constructs a gene targeting vector containing a mutation in the target gene; 2) the targeting vector is introduced into ES cells; 3) ES cell clones which undergo homologous recombination with the targeting vector are identified; 4) ES cell-mouse chimeras are produced; 5) the chimeras breed and transmit the chromosome with the targeted gene to their offspring; 6) homozygous animals are produced from the mating of hemizygous chimera offspring; and 7) the phenotype resulting from the genetic mutation is characterized. Core personnel are available for consultation on all aspects of gene targeting research. Contact Thom Saunders with any questions or Meet the Staff.  The Transgenic Core prioritizes all requests for service on a “first-come, first-serve” basis. Your requests for electroporation, clone expansion, and blastocyst microinjection will be added to the Transgenic Core work queue in the order that they are received.

2. Plan the experiment

This is a brief outline of the steps necessary to produce mice with a mutation targeted to a specific gene. These animals are referred to as “knock-out” mice or “gene targeted” mice. The broad outline of the experiment includes: 1) the investigator constructs a gene targeting vector containing a mutation in the target gene; 2) the targeting vector is introduced into ES cells; 3) ES cell clones which undergo homologous recombination with the targeting vector are identified; 4) ES cell-mouse chimeras are produced; 5) the chimeras breed and transmit the chromosome with the targeted gene to their offspring; 6) homozygous animals are produced from the mating of hemizygous chimera offspring; and 7) the phenotype resulting from the genetic mutation is characterized. Core personnel are available for consultation on all aspects of gene targeting research. Contact Thom Saunders with any questions or Meet the Staff.  The Transgenic Core prioritizes all requests for service on a “first-come, first-serve” basis. Your requests for electroporation, clone expansion, and blastocyst microinjection will be added to the Transgenic Core work queue in the order that they are received.

3. Clone and the Verify the Targeting Vector

This is a brief outline of the steps necessary to produce mice with a mutation targeted to a specific gene. These animals are referred to as “knock-out” mice or “gene targeted” mice. The broad outline of the experiment includes: 1) the investigator constructs a gene targeting vector containing a mutation in the target gene; 2) the targeting vector is introduced into ES cells; 3) ES cell clones which undergo homologous recombination with the targeting vector are identified; 4) ES cell-mouse chimeras are produced; 5) the chimeras breed and transmit the chromosome with the targeted gene to their offspring; 6) homozygous animals are produced from the mating of hemizygous chimera offspring; and 7) the phenotype resulting from the genetic mutation is characterized. Core personnel are available for consultation on all aspects of gene targeting research. Contact Thom Saunders with any questions or Meet the Staff.  The Transgenic Core prioritizes all requests for service on a “first-come, first-serve” basis. Your requests for electroporation, clone expansion, and blastocyst microinjection will be added to the Transgenic Core work queue in the order that they are received.

4. Electroporate ES Cells and Select Recombinants

ES cells are a very sensitive reagent, and most often the weakest link in the process of producing gene targeted mice. Fastidious tissue culture technique is necessary to maintain the pluripotent state of the cells during electroporation, selection, and expansion. In order to maximize successful outcomes, the Transgenic Core maintains ES cells and companion reagents that are quality tested by producing ES cell-mouse chimeras that transmit the ES cell haplotype to offspring. Investigators may choose to use the Core’s Gene Targeting Service (we do all of the tissue culture work, you do all of the molecular biology), or to use space in the Core lab (rent space in our completely equipped laboratory for ES cell culture work), or to do the tissue culture work in their own space. After the cells are electroporated with the targeting vector and put under drug selection, resistant clones are picked and cultured. DNA prepared from the clones is screened and ES cell clones which have undergone homologous recombination with the targeting vector are identified. During screening the clones are cryopreserved in 96 well plates at -80 degrees C. Since these conditions are not optimal for long term storage of cells, it is important to complete the screen in a timely fashion.

After clones are identified, they are brought out of the freeze and expanded. During expansion, additional DNA is prepared and tested, to confirm recombination with the targeting vector, clone morphology is examined, chromosome counts are used to see if the clone is euploid, and a mycoplasma test is done to see if the cells are infected. Investigators may choose to have the Core carry out these tasks, perform them in the Mouse ES Cell Laboratory, or to do them in their own space. In our experience we find that the identification and expansion of five gene targeted clones is enough to generate three euploid, targeted clones for blastocyst microinjection. Once targeted clones have been verified the next step is to…

5. Inject Blastocysts with ES Cells

The Core will microinject ES cells into blastocysts. See the service description for more information. The goal is to produce chimeric animals with high contribution from the ES cell clone and low contribution from the host embryo. This is typically assessed by coat color contribution. ES cells derived from 129 mouse substrains produce agouti fur. When these ES cells are microinjected into C57BL/6 blastocysts (black fur) the resulting mice will appear as agouti on black chimeras.  When ES cells are derived from C57BL/6 mouse subtrains the they are injected into albino C57BL/6 mice to produce black on white chimeras. ES cell-mouse chimeras with high coat color contribution from the ES cells are likely to transmit through the germline more quickly than low coat color contribution chimeras. Another sign that the ES cells successfully colonized the host embryo is a distorted male:female sex ratio in the chimeras. Since ES cells are X:Y, a good clone will convert female host embryos to phenotypic males. Thus, the desired outcome is for 75% or more of the chimeras to be male and to include animals that have 90% or more ES cell coat color contribution. Unfortunately, we can not guarantee that all ES cell clones will produce germline chimeras. In our experience we have seen very good clones that produce high contribution chimeras transmit to 100% of their offspring and we have also seen apparently “lethal” clones in which zero animals were born after transferring 90 injected embryos, we have seen “bad” clones in which no animals with  ES cell coat color were produced, we have seen “bad” clones which produced low ES cell coat color contribution chimeras. Unfortunately there are no in vitro tests that will differentiate among these outcomes, the only way to discover the germline potential of an ES cell is to inject it. In our experience we find that when three euploid, gene-targeted are used to make ES cell-mouse chimeras that at least one and often two of the clones will produce germline chimeras.

6. Chimera Breeding and Germline Transmission

Once the chimeras are produced, breeding is carried out to obtain offspring that carry the targeted gene. Male ES cell-mouse chimeras are mated with C57BL/6 females or albino C57BL/6 mice (also see Breeding Suggestions). The first sign of germline transmission is the appearance of pups with agouti coat color (129 mouse derived ES cell lines) or black coat color (C57BL/6 derived ES cell lines).. Pups produced from sperm derived from ES cells will have agouti coats or black fur.  Half of the animals should inherit the targeted gene. Pups that are produced from sperm derived from the C57BL/6 host embryo will have black coats and those from the albino C57BL/6 host embryo will be white. The Transgenic Facility will breed ES cell-mouse chimeras with the appropriate mouse strain for germline transmission on a fee for service basis.Tail biopsies from the pups are screened for the presence of the targeted gene in the same way that you screen for the presence of a conventional transgene.

7. Breeding and Analysis

Once hemizygous mutant mice are identified they are mated to produce homozygous mice. The final stage is to study the animals to determine the consequences of the mutation introduced by homologous recombination in ES cells.